This project's goal is to define alterations in proto-oncogenes and tumor suppressor genes that play a role in B6C3F1 mouse hepatocarcinogenesis. PCR amplification and DNA sequencing have been employed to characterize the activating mutations in nine unusual H- and K-ras proto-oncogenes of chemically induced B6C3F1 mouse hepatomas that lacked H-ras proto-oncogenes of chemically induced B6C3F1 mouse hepatomas that lacked H-ras 61st condon alterations. In each tumor CG- >GC transversion was observed at the 1st position of condon 13; this result is quite surprising based on predicted mutagenic specificity and the absence of any 12th condon ras mutations in over 200 hepatomas examined to date. The presence of a single oncogene in transgenic mice is generally insufficient for malignant transformation. Since B6C3F1 hepatomas frequently contain mutated H-ras genes, we asked whether ras activation is a secondary genetic even during hepatocarcinogenesis in transgenic mice carrying a SV40 large-T antigen/metallothionein enhancer construct (provided by A. Messing, U.W.). H-ras genes of these hepatomas were amplified by PCR and sequenced, but no mutations were detected; this study will be extended with additional transgenic constructs. Inactivation of tumor suppressor genes is another common genetic alteration in human cancers. This has been detected by loss of heterozygosity in specific chromosomal regions using restriction fragment length polymorphism (RFLP) analysis. We have extended these studies to DNA from chemically induced B6C3F1 hepatomas with several RFLP probes, including the retinoblastoma gene, but no losses of heterozygosity have been detected to date. In order to generate tumors in additional tissues for RFLP analyses a panel of transgenic mouse lines containing oncogenes under the control of various tissues specific transcriptional regulatory elements is being constructed in collaboration with M. Eddy and G. Goulding (LRDT). A collaboration has also been initiated with W. Caspary (CGTB) using a contract mechanism to generate and map a large number of new RFLP probes for B6C3F1 mice. NTP bioassay tumors from a variety of tissues will be screened with these probes.